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An Easy Test for Bacteria 
in Milk and Cream 

(Barthel's Reductase Test Improved) 



BY 



HUGO JONE 

Chief Chemist and Bacteriologist of the Empire State Dairy Co., 

Brooklyn, N. Y., late Chief Chemist of the Chicago 

Health Department. 



Price 50 Cents 

(Prepaid) 



HUGO JONK 
P. O. Box 37, Station A, Brooklyn, N. Y. 

1915 



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An Easy Test for Bacteria 
in Milk and Cream 

(Barthel's Reductase Test Improved) 



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BY 



HUGO JONE 

Chief Chemist and Bacteriologist of the Empire State Dairy Co., 

Brooklyn, N. Y., late Chief Chemist of the Chicago 

Health Department. 



Price 50 Cents 

(Prepaid) 



HUGO JONE 
P. O. Box 37, Station A, Brooklyn, N. Y. 

1915 * 



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ERRATA 



Page II. lilies ?i and 2^: The leords "It tlifre is any. the mixing was 
not done thoroughly." «sv to be omitted. 

Page II, line 2J : Tlie -tx'ords "and placed into a wire basket having a 
layer of cotton at the bottom" are to be omitted. 

Page ip, line u from the bottom: The ivords "If, in addition to using 
8 cc of blue solution, the air is excluded from the tubes as directed on 
page 24," are to be inserted so as to prceede the zeords: "the results obtained 
in this manner are quite accurate and may be relied on as indicating correctly 
the extent of the bacterial activity in the milk." 



/ 



JUL 12 1915 



INTRODUCTION. 

The problem of improving the sanitary quality of a milk supply is incapable of solu- 
tion so long as there is not a simple test available for determining approximately the bac- 
terial contents in the country. No amount of testing done in the city, at the point of 
consumption, can ever prevent the most serious pollution of the milk, which is that oc- 
curring previously, in the country, at the point of production. Much of the testing must 
be done in the country. If it is omitted, the creamery manager does not know which of 
the farmers that furnish milk to him, is to be held responsible whenever the bacteria run 
high in the mixed milk which he ships to the city. Before the cause of pollution can be 
removed, it must of necessity first be located. This cannot easily be done without a test. 
It is true that the inspectors sent out by the creameries to score the dairies discover 
some causes of pollution. But the inspectors need a test to determine beforehand which 
of their numerous dairies require inspection the most. Furthermore, experience shows 
that the inspectors are frequently deceived by the farmers. A farmer may have his dairy 
in first class condition when the inspector is around and yet deliver milk which runs ex- 
cessively high in bacteria, without being discovered by the inspector. He may, for in- 
stance, feed sour ensilage during the milking, or keep his evening's milk at 75° F. instead 
of 45° F. over night in summer. So long as a few pieces of ice are put between the cans 
just before delivery, the inspector is easily cheated. His thermometer certainly fails to 
detect the excessive number of bacteria in such milk. The color test is the only practical 
way to locate the trouble. Actual experience proves this. 

Over ten thousand samples of milk were tested, at receiving stations in the country, 
by the color test after my directions, during a period covering two years. These tests 
have been so successful for the purpose of locating the causes of high bacteria that milk 
dealers who had not used the test, but who had heard of it, quickly adopted the color test 
for their own creameries. 

Furthermore, well known sanitarians and authorities on milk hygiene made inquiries 
concerning the test, as improved by me. 

Under these conditions, I am sure I am complying with a general demand in pub- 
lishing a detailed description of the test as improved, and directions for making it. 

These directions, as will be noted, are so specific and so simple that every 
creamery man, including those who never had any previous experience in laboratory 
work, will be able to make the test without further instructions. 

To the bacteriologists and chemists who may read my directions, I beg to apologize 
for using so simple a language and making statements of facts which are most elemen- 
tary and well known to them. As thousands of creamerymen, including those who are 
without experience, are expected to use my directions, I believe I am right in giving 
elementary instructions, even at the risk of making those directions tiresome to the pro- 
fessional laboratory men. As the latter are comparatively few in number, the harm 
done by excessive simplicity will not be serious. 

It will be noticed that this publication is designed to meet the needs of the milk 
dealers, creamery employees, condensary employees and others interested, not only as 
regards contents, but also as regards form. 

My statement that the color test, or reductase test, is the only practical test for 
locating the cause of high bacteria in the country, is amply corroborated by authorities 
whose judgment cannot be questioned. For instance. Dr. H. C. Campbell of the patho- 



logical division of the U. S. Bureau of Animal Industry, an expert in milk hygiene, in 
speaking of the reductase, or reduction test and other biochemic tests, in the annual 
report of the bureau, for 1911, after experimenting with them, comments on these tests 
as follows: 

"The reduction test agreed in most instances with the bacterial count ; that is, when- 
ever the sample was reduced in a short time, the bacterial count was high." 

"The biochemic tests used are simple in their technique. The results obtained are 
practically as good as those shown by plating, so far as estimating the number of bac- 
teria is concerned." 

"These tests, if applied at the dairy or site of milk production, would greatly aid the 
health officials in controlling a milk supply." 

"In all pathological processes of the udder the reductase properties are increased." 

Dr. Wm. Ernst, director of the milk control station at Munich; Dr. John R. Mohler, 
chief of the pathological division, U. S. Bureau of Animal Industry, and Dr. A. Eichhorn, 
senior bacteriologist of the same division, in their "Text Book of Milk Hygiene", pub- 
lished in 1915, make the following statements regarding the reductase test: 

"Very valuable results are obtained with the reductase test when conducted in con- 
nection with the microscopic examination of the sediment." 

"As already mentioned, for practical control work the counting of bacteria may be 
omitted. In this work the reductase test offers a quicker determination of the spoiled 
condition of the milk." 

"... If (in Miiller's experiments) sour milk or cow manure was added to fresh milk, 
the time of reduction was hastened. He therefore proved that all factors which favor- 
ably influence bacterial growth, hasten the reduction." 

"The methylene reductase test gives very good information relative to bacterial 
multiplication." 

Manifestly, the extraordinary usefulness of the reductase test is recognized by the 
leading authorities on milk hygiene. There is a pressing need for a simple, easy, and 
rapid test for determining the sanitary quality of milk at all creameries, condensaries, 
dairies and similar places in the country, and the reductase test answers the purpose 
much better than any other test in existence. 

In sharp contrast with the favorable comment of the experienced laboratory men is 
the comment which comes from superficial and inexperienced observers. Young students 
and others who had little or no experience in laboratory work, naturally failed to get 
correct results, chiefly because they did not prepare the methylene blue solution prop- 
erly. Their technique was faulty because they could not devise their own technique,, 
while the published directions failed to mention the necessary points of technique in 
making the solution and the test. Their failure to get correct results has evidently no 
weight in judging the merits of the test. The test can certainly not be held responsible 
for the mistakes of beginners who misused it at a time when elementary instructions for 
making it were not yet available. This does not say, however, that the test could not 
be improved in other respects. Both, the instructions and the test needed improvement. 
The proper thing to do was evidently to give elementary instructions for making the 
test. This is what I have undertaken to do. The test itself needed improvement as 
regards the amount of methylene blue used. In this respect, criticism of the old form 
of the test hitherto in use may have been justifiable to a certain extent. Realizing that 
the reductase test is the only test in existence which is simple enough to be used in the 
country, and that it was badly needed, I undertook to improve it, and the thousands of 
results obtained with the improved form show that I was more or less successful. 



It may be surmised that the improvements I made on the test are superfluous be- 
cause experienced laboratory men could get satisfactory results without such improve- 
ments. This is, however, not the case. For instance, without a chemical standardiza- 
tion, the strength of the methylene blue solution was bound to vary, even in the hands 
of an expert, so long as it was based on saturation in alcohol or a similar solvent. A 
saturated solution made in summer is apt to be stronger than one made in winter, since 
warm alcohol dissolves more methylene blue than cold alcohol. Furthermore, the ad- 
mixture of foreign substances sometimes found in methylene blue may also produce a 
variation. The chemical standardization is therefore necessary even if errors in technique 
are avoided. It secures more accurate results. 

The "Double Time Test" and the bacteriological regulation of the amount of methy- 
lene blue used will also be found helpful in obtaining more accurate results. 

Thus the improvements and the elementary directions will enable both, the experts 
and the beginners, to get better results hereafter. 

The plating test with agar is a failure in the country for more reasons than one. 
Aside from the excessive first expense for the complex equipment used in this test, the 
current expenses are high for the reason that only a professional laboratory man can be 
relied on as furnishing correct results. Beginners who believe the results they obtain to 
be correct, are nearly always mistaken. The test has sometimes failed to give correct 
results in the hands even of the most expert bacteriologists, as Dr. H. W. Conn, the 
well known "specialist in agricultural bacteriology, has shown. This test is even more in 
need of an improvement than the reductase test had been. With all the money spent 
on this test, little is accomplished. 

In most creameries it would be impossible anyway to use the test successfully even 
for a professional bacteriologist, since the equipment is never complete. It is for in- 
stance difficult to see how an incubator could be kept at constant temperature in a village 
where neither gas nor an electric current is available. An alcohol lamp would have to be 
used the same as in the case of the reductase test, with this difference, however, that it 
is practical to use it in day time for the reductase test, but quite impractical to use it at 
night for the incubator. 

Even this is not yet the last of the disadvantages connected with the plating test. 
If the creamerymen had to wait 48 hours for the results, as in the plating test, they 
would fail to locate the causes of those pollutions which disappear in less than that 
time. They could not reject polluted milk delivered to them on the strength of the plat- 
ing test, since the milk has to be either rejected or accepted in much less time than 48 
hours. 

Furthermore, the plating test lost much of its former value as an index of the sani- 
tary quality of raw milk from the time on that pasteurization of milk became general. 
As the bacteria are killed anyway by pasteurization, the real pollution in the finished 
pasteurized milk does not consist in bacteria, but in the metabolic compounds left behind 
by the bacteria. The amount of these metabolic compounds should be determined by 
exact methods of quantitative chemical analysis and not by any of the biological 
methods, each of which is more or less inexact. The number of live bacteria, even if it 
were determined accurately, fails to measure the amount of metabolic compounds cor- 
rectly for the further rea.son that these compounds are largely derived from the dead 
bacteria which in winter are so numerous, and which, evidently, do not show up at all 
in the plating test. The number of live bacteria, in other words, is out of proportion 
with the real pollution. 



I do not see how any intelligent manager or director of a milk company can seriously 
consider the plating test for general adoption in the country stations. I know well that 
it is desirable to use the same test which the officials use. However, the days of the 
plating test, as the exclusive official test for raw milk to be pasteurized, are numbered. 

For that insignificant portion of market milk which is sold raw to the consumer, the 
plating test may remain a valuable index of the sanitary quality, but not for raw milk 
to be pasteurized. Conscientious officials will admit that the plating test is not fit to be 
made the sole basis of legal prosecution for such milk, since it is misleading in several 
ways. 

If the number of spore bearers is to be determined, the plating test again fails to 
give correct results, since the ratio of the spore bearers to the total number of live bac- 
teria fluctuates constantly. 

The direct microscopic count of the bacteria in milk after Skar's or Brew's method 
is a little more accurate than the plating test. It cannot be made entirely accurate, 
chiefly because only an extremely small amount, viz.: much less than a drop, can be 
examined. Such a small amount gives inaccurate results for the reason that it does not 
represent the number of bacteria in the milk supply correctly. 

Furthermore, the microscopic test, like the plating test, is expensive and requires 
the services of a professional laboratory man. A microscope costing at least $75.00 can- 
not be turned over to a creameryman who has had no previous experience in such work. 
The time may come when every creamery manager will be required by law to pass an 
examination in bacteriology, chemistry, and milk hygiene before he is allowed to put 
up milk for infants, just as a pharmacist has to pass an examination in materia medica, 
chemistry, and dispensing before he is allowed to put up prescriptions. Such laws, in fact, 
are already in force in certain sections in Europe. For the present, however, we must 
take the creamerymen as they are and we cannot expect them to do scientific work of a 
high order, or at least not all of them. Therefore we must use a simple test, such as the 
reductase test, for the next twenty or thirty years to come, for this reason, and prob- 
ably thereafter for other reasons. 

The reductase test measures accurately the extent of bacterial activity, in the 
"Double Time Test," if the exact length of time required for decolorization is deter- 
mined. This is a more valuable criterion to go by, in judging raw milk to be pas- 
teurized, than the mere number of bacteria, since it shows the capacity of the bacteria 
for decomposing milk and for forming harmful decomposition products. Furthermore, 
the fact that hydrogen, hydrogen sulphid, and mcrcaptans generated by such organisms 
as the members of the coli-aerogenes group and the bacteria of putrefaction, decolorize 
the methylene blue in the reductase test, plainly shows that the reductase test indicates 
harmful decomposition, besides the mere extent of decomposition. That certain diseased 
conditions of the udder can be detected by the reductase test, is known. Under these 
circumstances, I am satisfied that the reductase test is a much more valuable index of 
the sanitary quality of raw milk to be pasteurized than the plating test. If the results 
of the plating test are in conflict with those of the reductase test properly carried out, 
it is merely one of the numerous confirmations of the fact that the plating test is un- 
reliable as an index of the sanitary quality of such milk. 

On agar, the milk bacteria can never de\-clnp those decomposition products which 
I hey would develop in milk. Consequently it is useless to employ agar as a culture 
medium if the nature and amount of those decom])osition products are to be determined, 
as in the case of raw milk to be pasteurized. The only proper medium for milk bacteria 
is milk, in such a case. This is another reason why the reductase test is valuable. 



The reductase test never fails to detect any lack of cooling in summer, in raw milk. 
It also detects any admixture of dirt. To determine whether a rapid decolorization is 
due to lack of cooling or to dirt, the acidity test is made. If the acidity is considerably 
above normal, therefore considerably above 0.17 per cent, the rapid decolorization is due 
to lack of cooling. If not. it is due to dirt. It is understood, however, that these tests 
are made on fresh milk. If there is an interruption of transportation facilities and the 
milk is kept in the country several days, it is apt to show a high number of bacteria and 
a rapid decolorization when there was neither dirt nor lack of cooling. These abnormal 
and rare conditions are to be taken into consideration, if they occur, in distinguishing 
between lack of cooling and dirt, as above stated. 

For pasteurized milk and pasteurized cream the reductase test is not recommended. 
The heat of pasteurization interferes with the test. 

Cheese makers and others who desire to make the fermentation test will find that 
the apparatus furnished for the reductase test can just as well be used for the fermen- 
tation test, the only difference being that the blue solution is omitted and the tem- 
perature is lower, viz: 98° F. or 75° F., as may be desired. 

It will be found convenient to have the standard methylene blue solution made in 
the city and to send it to the milk-receiving stations in the country. Likewise, any bac- 
teriological tests for the purpose of determining how much of the standard solution is 
to be used for a given milk supply, may conveniently be made in the city. It will be 
noticed that the scientific work may easily be separated from the elementary work of 
making the tests on the milk samples, as the solution made in the city or the results of 
the plating test can easily be sent anywhere. This enables the creamerymen to get all 
the accurate results which scientific laboratory work may afiford, without having to do 
the scientific part of the work themselves. Under these circumstances anybody can 
make the test in the country and expensive help is unnecessary, so far as the country 
is concerned. 

It is obvious that the reductase test affords the greatest advantages in summer. 
Whenever the number of bacteria is excessive either from lack of cooling, or from dirt, or 
from any other cause, this test never fails to detect it. Milk dealers who are shipping milk 
to cities or towns where the number of bacteria is limited by law, will have no trouble 
in locating, by this test, those farmers whose milk exceeds the limit. This will enable 
them to live up to the bacterial limits in the cities and towns, since they can exclude 
the objectionable milk from their shipments. ' 

The results of the reductase test generally agree better with the plating results in 
summer than in winter. 

Officials would do well to adopt the reductase test as the official test. They cannot 
expect the milk dealers to use the plating test, since it is impractical and misleading. 
Some kind of a test will have to be adopted because without such a test, the milk dealers 
cannot live up to bacterial standards. 

Creamerymen who ship milk to New York City arc reminded that the present limit 
(1915) for Grade B milk, raw, is lyi million bacteria per cubic centimeter, if the milk 
is to be pasteurized in the city. This corresponds to three hours in the "Double Time 
Test," just as it corresponds to 1^^ hours in the "Single Time Test." 

All creamerymen and others making the test arc urged to study those sections in 
the pamphlet which arc printed in bold type, and to post the tables which go with the 
pamphlet in the testing room for daily use. 



The color of the tubes should be observed in bright daylight, not at gas light or 
other artificial light, and the blank tube for comparison should not be omitted. The 
color can be seen best on a white background. 

Bacteriologists who are called upon to regulate the amount of the standard methy- 
lene blue solution in accordance with their plating results, are reminded that the "Single 
Time Test" and the "Double Time Test" want to be regulated each independently of the 
other. It is not safe merely to use five times as much blue solution for the "Doube Time 
Test" as was found necessary for the "Single Time Test." Each test must be compared 
with plating results directly. 

The reductase test, as improved, will be found to give correct results. In summer, 
the results agree so well with the plating results that the plating test itself, if it were 
made in the country, could not furnish any better results. While excessive numbers of 
bacteria are always detected, a moderate number of bacteria can escape detection only, 
if the test is not performed properly. Generally, whenever an inaccurate result is ob- 
tained, it is due to insufficient mixing of the milk sample. It is to be remembered that 
only a very violent agitation of the milk will break up the clumps of bacteria. If the 
sample is placed in a sterilized bottle, half filled, and the bottle shaken very vigorously 
25 or 30 times, correct results are obtained every time. Can milk must be stirred vigor- 
ously before sampling. 

Bacteriologists who compare plating results with the results of the reductase test, 
will find it convenient to use for the milk samples sterilized bottles of about six ounces 
capacity. Having the bottle only half filled, to make shaking easy, a good representa- 
tive and well mixed sample is secured for both tests, and enough is left to repeat the 
tests, if necessary. 

As the color test affords an easy means for detecting excessive numbers of bac- 
teria, milk dealers need no longer run the risk of having their milk condemned by in- 
spectors and of having their reputation ruined. If the color test shows that a given can 
of milk is above the legal limit for bacteria, all they have to do, is to send it to the 
churn instead of selling it as sweet milk. Or better still, they can make a contract with 
their farmers specifying that the milk furnished to them must not be decolorized, by the 
"Double Time Test," in less than 2^/2 hours, or 3 hours, or 4 hours, as the case may be. 
As they are then at liberty to return the milk to the farmer, without loss to them, if it 
runs high in bacteria, they can make sure that their milk will always pass inspection. 
Milk dealers, in other words, need not dread the consequences of official inspection any 
longer. 



JUL 12 1915 



A 



Barthel's Reductase Test 

Improved by Hugo Jone 



Directions for Making the Color Test (Methylene Blue Test) 

for Bacteria in Milk 



Copyright 1915 by Hugo Jone 



Most bacteria living in milk separate an enzym or body juice known scientifically 
by the name of reductase. The reductase has the property of destroying the blue color 
of the aniline dye known as methylene blue. If there are many millions of bacteria in a 
sample of milk, the large amount of reductase they separate is sufficient to decolorize at 
once the measured amount of methylene blue solution usually added to the milk for the 
test. If there are few bacteria, they do not separate enough reductase for decolorizing 
the methylene blue completely. However, if the bacteria are allowed to multiply by 
putting the milk in a warm place (usually 112' Fahrenheit), they will be so numerous in 
a few hours, that the reductase they separate is finally sufficient to decolorize the methy- 
lene blue completely. Manifestly, under these circumstances, a milk sample having many 
millions of bacteria in it, will be decolorized rapidly, while some samples having at the 
start only a few thousand bacteria in it, will require several hours for decolorization. 
Thus, we can tell the number of bacteria by the time required for decolorization. The 
samples must be kept at 112° F. A convenient apparatus for keeping the samples warm 
is a double boiler provided with an alcohol lamp and a tube holder. The tubes with the 
samples remain in the warm water until decolorized. The inner pot of the double boiler 
may be used alone. 

9 



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(1) The inner pot is seen standing on the tripod, with a test tube holder, test tubes, 
and a bulb thermometer on the inside of it. 

(2) The outer pot is seen standing on the table. 

(3) The methylene blue solution in use is contained in the wide-mouth bottle near 
the corner of the table next to the glass cap. (4) The glass cap is taken off only for the 
purpose of making both, the bottle and the cap, better visible. In the actual testing, the 
cap is never removed from the bottle except at the moment when solution is taken out 
of the bottle with the pipette. All dust is to be kept out of the blue solution that way. 
This style bottle is called an English oil bottle. It is especially well adapted for holding 
a pipette besides a liquid without polluting either. 

(5) The wire basket with empty tubes, (6) the glass tumbler with decolorized tubes, 
(7) the pint bottle with mushroom glass stopper containing methylene blue solution 
ready for shipment, and other objects will be recognized without further specification. 

The pipette fitted into the English oil bottle is a short nozzle pipette measuring 
2 cubic centimeters of blue solution. 

The other pipette (8) shown standing in one of the glass tumblers is a short nozzle 
pipette measuring 10 cubic centimeters of milk. As 20 cubic centimeters of milk are used 
for each test, the pipette must be dipped into the milk twice, instead of once, when 
sampling. This is done on purpose to secure a good average sample of the milk. 



10 



The test is made as follows : — Enough warm water is placed in the outer pot of the 
double boiler to nearly fill the jacket formed when the inner pot is put inside, to insure 
uniform heating of the inside pot. Furthermore, in the inner pot enough water is put to 
submerge completely the milk samples in the test tubes, thereby insuring uniform heat- 
ing of the milk samples. It would not do to have the milk in the tube stand higher than 
the surrounding water, because that portion of the milk column which sticks out above 
the water would cool off too much and in that way require more time than it should for 
decolorization. 

The water in the inner as well as in the outer pot is brought to a temperature of 
112 F. The water in both pots is then continued at 112 F. by means of the alcohol 
lamp. 

The test tubes which contain 20 cubic centimeters of the different milk samples, 
each, and which are identified by the numbers on them, are then put into the water until 
they are as warm as the water, which is 112 F. Then 2 cubic centimeters of the blue 
solution are dropped into each tube and mixed very thoroughly with the milk sample. 
A rubber stopper may be used to close the tube, to facilitate mixing. This can be 
cleaned by wiping it with a clean towel. However, the rubber stopper must be cleaned 
each time after use. If the towel is not strictly clean, it may spoil the succeeding tests. 
After mixing, each tube is closed with a cotton stopper. 

After dropping the 2 cubic centimeters of blue solution into each of the tubes, the 
time is noted down, the color is observed, at fixed intervals thereafter, and the number 
of bacteria estimated by the time required for complete decolorization. The blue color 
must be turned into a plain white completely, except that at the top a faint blue ring 
may remain. There must be absolutely no blue color left at the bottom. If there is any, 
the mixing was not done thoroughly. 

When observing the color of the tubes, they may be lifted out of the warm heater 
and placed into a wire basket having a layer of cotton at the bottom. However, they 
must be put back into the water immediately to prevent their cooling down too much. 

The number of bacteria is estimated as follows: If less than ' i hour is required for 
decolorization, over 5 million bacteria, if ■ _. hour, 3 to 5 million, if '4 hour, 2 to 3 million, 
if V/2 hours, 1 to 2 million, if 2'j hours, 300,000 to 1 million, if 3'j hours, 100,000 to 
300,000, if over 4 hours, less than 200,000. 

The tubes must be rinsed and then soaked in boiling hot water for at least 15 minutes 
after each use. If any milk adheres to a cotton stopper, it must be soaked off or washed 
off with hot water and the stopper dried thereafter. 

Great care should be taken to keep the tubes at the proper temperature during the 
test. It must be 112 F. A deviation of as much as 5 degrees will influence the result 
perceptibly. The color must never be dropped in until the temperature of the milk is 
112 F. or nearly so. If the milk is still cold, the time required for the decolorization 
will be unduly increased. 

For the cotton stoppers, non-absorbent cotton should be used. It keeps clean longer 
than the absorbent cotton. 

In testing high grade milk which is intended for infant feeding, it is generally neces- 
sary to keep the tubes in the warm water about twice as long as in the case of common 
market milk, since it takes longer to decolorize the clean milk. 

The Health Department of New York City allows not more than 200,000 bacteria 
(determined by plating on agar) per cc in infant milk, or Grade A Milk, before pasteuri- 
zation. Therefore this milk must remain blue at least 4 hours. It must, however, be 
remembered that this test gives only approximate results, and is not as yet official. 

11 



For Grade B Milk before pasteurization, the Health Department allows not more 
than 13/2 million bacteria per cc. Therefore this milk must remain blue at least 
lyo hours. 

The strength of the methylene blue solution is of the utmost importance for the cor- 
rectness of the test. A strong solution certainly requires more time to decolorize than a 
weak solution. Therfore, it must always have the exact strength, not more and not less. 
The testing and adjusting of the strength of the solution is to be left to a professional 
chemist, it being too technical for others. The strength of the solution is so regulated 
that the results obtained by the color test are about the same as those obtained by 
plating on agar. If they are not the same, the strength of the solution is corrected until 
they are the same. 

The bottles containing the methylene blue solution must never be filled to the top, 
but only half iilled. The solution keeps better in contact with air. Therefore, there must 
be an airspace in each bottle. 

The time required for decolorization is to be reported by one of the following time 
limits : — 

Less than J4 hour. 
yi hour 
^ hour 
IJ^ hours 
2]/! hours 
3^4 hours 
over 4 hours. 

In heating the apparatus with a lamp, only denatured alcohol or wood alcohol is to 
be used for burning. Never use gasoline, as it is liable to explode. 

If the flame of the alcohol lamp flickers and the heating is irregular, the outside 
pot, if used, will maintain a fairly steady temperature in spite of the irregular heating. 
If on the other hand, the alcohol flame is protected from draught and not allowed to be 
extinguished or to flicker, the outside pot may be omitted, and the inside pot heated 
directly by the flame, in summer. 

If in winter, the testing is done in a cold room, it is again desirable to use the out- 
side pot besides the inside pot, for the reason that the water near the circumference of the 
inside pot would cool down rapidly and produce inaccurate results, if no jacket were 
used to keep the inside pot warm all over. 

It will thus be seen that the outside pot can be eliminated only by regulating the 
flame, and in winter also the temperature of the room. 

The outside pot may be used as a receptacle for the tubes when they are sterilized 
with hot water, at times when it is not needed as a jacket in the apparatus. 

Before taking the samples of milk for the color test, the milk must be stirred 
sufficiently to mix the cream uniformly with the rest of the milk. If this is not done, 
too much cream goes into the samples and the results obtained are too high, as the cream 
contains more bacteria than the thin milk. Therefore, be sure to stir before sampling. 

All tests should be made immediately after the samples are taken. If the samples 
are kept in a warm place before testing for more than half an hour, they will decolorize 
quicker and give incorrect results. 

Whenever the capped bottle is filled with new solution, the remnant of the old 
solution in the bottle is to be wasted. It must not be mixed with the new solution. 

12 



' y 

A40T«r^ V g^j.j]^el'g Reductase Test 

%o I Improved by Hugo Jone 

PART II / 

An Improved Methylene Blue Solution for the Reductase 
Test and Some Modified Forms of the Test 



Copyright 1915 by Hugo Jone '^ 



The strength of the methylene blue solution used in the reductase test for the de- 
termination of the number of bacteria in milk, has in the past not received as much 
attention as it needs. Some of the failures to obtain correct results were due to the 
lack of a suitable apparatus, others were due to the application of European methods to 
American milk supplies without the necessary adaptation of those methods to American 
conditions, but most of the failures were due to the use of solutions which were either 
too dilute or too concentrated. 

Confusion was caused by the use of different brands of methylene blue. Neither the 
make nor the label was specified. The alcoholic solution was sometimes fully saturated 
and sometimes only half saturated. Sometimes this solution was merely decanted or 
pipetted oflf, instead of being filtered, with the result that particles of solid methylene blue 
entered the diluted aqueous solution, making it far too strong. The moisture content 
of the alcohol varied, and the strength of the saturated alcoholic solution varied with it. 
Nearly every one of the experimenters used a solution of different strength, and yet, 
they were surprised when they obtained different results. 

In addition to this, there were the differences in the milk supplies tested, such as 
are brought about by the seasons and by the differences in climate. As the production 
of reductase is associated chiefly with organisms growing well in summer heat, it is 
evident that we cannot possibly expect the same solution which gives correct results 
in summer, to give correct results in winter also. Yet some experimenters have applied 
the summer solution to winter milk or the winter solution to summer milk in the vain 
hope of getting correct results. 

These difficulties with the variations in the nature of the milk and in the strength 
of the solution are certainly more than sufficient to account for all the discrepancies 
which ever existed between the results obtained by the reductase test. Furthermore, 
they explain why this test has been so often and so unjustly denounced as unreliable. 
Before anybody declares the test to be unrel'iable, he certainly wants to determine first 
whether his own work is not unreliable. 

13 



Neisser, Wechsberg, and Barthel deserve great credit for introducing the reductase 
reaction in practical milk testing, but they failed to put their method in a form which 
would enable the inexperienced operator to use it correctly. 

All the difficulties with the strength of the solution may be overcome by preparing 
and standardizing it in the following manner : 

PREPARATION. 

About 10 grams of that brand of methylene blue which is labeled "Methylen Blau 
fiir Bac, Koch" and which is made by G. Griibler, Leipzic, are mixed with about 600 cc 
of deodorized alcohol of the kind known as "Cologne spirits." The mixture is allowed 
to digest for twenty hours or longer at the ordinary room temperature. No harm is done 
if it is digested many months. It is advisable to shake the mixture from time to time 
to facilitate saturation. Just before using the mixture, however, it should be settled. 
150 cc of the clear, supernatant fluid are filtered, while the rest of the solution is retained 
for future use. 125 cc of the filtrate, which must be perfectly clear and free from par- 
ticles, are mixed with 275 cc of Cologne spirits, and then the alcoholic mixture is mixed 
with enough distilled water to make ten liters. In other words, the diluted solution is 
made up as follows : 

Alcoholic methylene blue solution, filtered 123.0 cc 

Cologne spirits 275.0 cc 

Distilled water, enough to make 10.000.0 cc 

This solution must be standardized before it can be used for the reductase test. 

THE BACTERIOLOGICAL STANDARDIZATION. 

A few tests are made with this solution on samples of raw milk which are also 
tested by plating on agar. As the solution is still more concentrated than it is intended 
to be, the results obtained with the solution will, in most cases, be lower than the num- 
ber of bacteria as determined by plating on agar. The solution must now be gradually 
diluted with distilled water until the results of the color test, when repeated, are about 
the same as those obtained by plating on agar. This correction of the strength of the 
methylene blue solution in accordance with the results obtained by plating is of the 
greatest importance for the success of the color test. 

It is desirable to select raw milk from different sources for comparing the two tests. 

A convenient way of making the comparison of the two tests is that of using the 
color test as a preliminary test for the plating. I need not point out the great need for 
a rapid test to indicate approximately the number of bacteria in the milk samples to be 
plated. Every bacteriologist knows that his results are incorrect if based on a plate 
having anything like a thousand or more colonies on it and that it saves him the making 
of three or four duplicate plates if he knows beforehand the approximate number of 
bacteria in each sample of milk to be plated. This advantage he secures by making, 
previous to the plating, the color test on the milk samples to be plated. In other words, 
he can determine what dilution to use in plating, by means of the color test. In less 
than three hours time, the color test will show him which of the samples contain an 
excessive number of bacteria and which, therefore, require a high dilution in plating. 

By using the color test in this way, the additional advantage is secured of checking 
the strength of the methylene blue solution by the plating results. If the solution is 
found to give too low results, it is evident that the solution is too concentrated, as it 
generallv will be when first made. Accordingly, as much distilled water is added to 
the solution as is thought necessary to have it give the desired results, or a little less 

14 



water. Too hasty addition of water is to be avoided, because the solution would be 
worthless if it should become too dilute. If, upon repeating the comparative tests on 
similar samples, the solution is found to give results which are still too low, more dis- 
tilled water is added and so on until it finally gives results which agree with the plating 
results. The strength of the solution thus found, is the correct strength to be used in 
testing milk supplies of a similar character. 

After the methylene blue solution has thus been standardized, it will be found to 
give correct results on all similar samples of milk without any further correction of its 
strength. The color test can then be relied on as giving approximately the number of 
bacteria, no matter whether it is checked by plating or not. 

Owing to this important advantage secured by the bacteriological standardization 
of the solution, this standardization should never be omitted whenever a new milk 
supply is tested the first time with the methylene blue. Thereafter it may be omitted so 
long as there is no material change in the nature of that milk supply. All that is neces- 
sary to do, is to leave the strength of the methylene blue solution unchanged. 

If, however, the milk supply undergoes vital changes in its jjacteriological contents, 
the solution nnist be re-standardized l^y comparison with plating results. 

In the fall of the year, for instance, the decreased number of reductase producing 
organisms on the one hand, and the increased number of dead bacteria on the other hand, 
necessitate a re-adjustment of the strength of the solution. Correct results are again 
obtainable after the strength of the solution has been re-adjusted to conform to the new 
conditions. 

It is evident that for similar reasons, a methylene blue solution which was fotnid 
to give correct results on milk produced in Canada, cannot be expected to give correct 
results on milk produced in Mexico, until it is re-standardized by comparison with 
plating results. 

It will be noticed that the sources of error which in the past have caused so much 
confusion and which have unjustly discredited the reductase test, are eliminated largely, 
if not entirely, by the proper preparation and the bacteriological standardization of the 
methvlene blue solution. Not only is the strength of the solution regulated, but it is also 
regulated in a way which makes it impossible for changes in climate, or season, or 
locality to produce errors in the test. Likewise, the trifling amount of reductase which 
may occur in fresh milk immediately after milking and which was declared by some 
people to render the reductase test "worthless", can evidently not produce any material 
error in the results, since the amount of reductase in fresh milk is appro.ximately con- 
stant, and since allowance is made for it in standardizing the solution. 

It ma^v" be argued that diflferent species of bacteria might produce different quan- 
tities of reductase and in that way influence the test. There is no evidence to show 
that those few species of bacteria which preponderate in milk, and which alone are 
numerous enough to influence the test perceptibly, differ materially in their reductase- 
producing capacity. The theory that dift'erent species might account for dift'erent 
results, was conceived for the purpose of explaining discrepancies which were in reality 
due to entirely different causes. It is to be remembered that the plating test itself is 
highly unreliable in its present form, as Conn has shown. It is therefore a poor criterion 
to go by. 

It is understood that, if a magnifier is used to count the colonies when standardizing 
the methvlene blue solution by plating, such a magnifier must also be used afterwards 
if the results obtained by the color test are to be checked by plating. Likewise, the agar 
should be the same both times, and the metliod of diluting also. 

15 



After the proper strength of the methylene bhie solution has once been determined 
for a given milk supply, at a certain season, it is not necessary to repeat the bacterio- 
logical standardization each year when that season begins again. All that is necessary, 
is to use a solution of the same strength as was used the preceding year for the same 
season. In order to fix and continue the exact strength of the methylene blue solution, 
a chemical method is used which determines the strength of the solution with mathe- 
matical accuracy. The strength of the solution is expressed in numbers of cubic centi- 
meters used up in a certain titration and the next year it is an easy matter for a chemist 
to make a solution of exactly the same strength even if he did not keep a sample of the 
solution from the preceding year. In this way, it is unnecessary to repeat the bacterio- 
logical standardization in all cases where milk supplies are to be tested which resemble 
each other as regards the season, or climate, or locality in which they were produced. 
A solution of the same strength, as determined by the chemical test, is used on all milk 
supplies which are known to be similar. 

THE CHEMICAL STANDARDIZATION. 

The strength of the methylene blue solution may be measured accurately by tritrat- 
ing it against a standard solution of stannous chlorid containing free hydrochloric acid. 
The blue solution is decolorized almost instantly when it touches the solution of stannous 
chlorid. This decolorization is analogous to the decolorization of the same solution in 
milk by reductase. It is due to the absorption, by the methylene blue, of hydrogen, and 
the formation of the leuco-compound, which is colorless. In the case of the stannous 
chlorid solution, the hydrogen is furnished by the breaking up of the hydrochloric acid 
molecule, while the chlorine combines with the stannous chlorid, forming stannic chlorid. 

As soon as the whole of the stannous chlorid is converted into stannic chlorid, the 
blue color re-appears and remains permanent. Thus, the blue solution is its own indi- 
cator, which simplifies the titration. It is evident that the strength of the methylene 
blue solution can be measured by the amount of stannous chlorid oxidized, since the 
amount of hydrogen absorbed by the methylene blue is equivalent to the stannous chlorid 
oxidized. 

In carrying out the titration, 11.8 milligrams of c. p. metallic tin in fine powder are 
dissolved in about 10.0 cc of concentrated, c. p. hydrochloric acid with complete ex- 
clusion of atmospheric oxygen. A few crystals of carbonate of soda are dropped into 
the liquid contained in a 300.0 cc Erlenmeyer flask which is closed by a rubberstopper 
carrying an india-rubber slit valve, also called "Bunsen valve." Manifestly the carbon 
di-oxid can pass outside, but the outside air cannot pass inside. 

The dissolving of the tin may be hastened by putting the flask in a warm place. It 
must be watched to make sure that the whole of the tin is dissolved. When the solution 
is complete, the liquid is diluted with a suitable quantity of recently boiled distilled 
water (about 20 cc, free from oxygen) and the blue solution run in from a burette until 
the blue color just begins to remain permanent. During the titration the atmospheric 
oxygen is again kept away by dropping in a few more crystals of sodium carbonate 
when first opening the flask. 

The volume of the methjdene blue solution consumed in the titration with the 
stannous chlorid formed from 11.8 milligrams of tin, amounts generally to about 
100.0 cc. However, as some milk supplies require strong methylene blue solutions, 
while others require weak solutions, as found by comparison with the plating results, the 
volume of blue solution consumed may be as low as 60.0 cc or as high as 1.^0.0 cc, for 
11.8 milligrams of tin each. 

16 



Chemists who keep a standard dichromate solution on hand, such as is used for the 
iodin number on fats or for the titration of ferrous salts, will find it convenient to pre- 
pare their stannous chlorid solution from the dry salt instead of dissolving the metallic 
tin, and to standardize the solution with their dichromate solution, using, if desired, 
potassium iodid and starch paste as indicator. A solution of suitable strength is ob- 
tained by dissolving about 100 milligrams of dry c. p. stannous chlorid in about 200.0 cc 
of distilled water strongly acidulated with hydrochloric acid. The strength of the me- 
thylene blue solution in this case may easily be calculated from the amount of the 
metallic iron which had been consumed in checking the strength of the dichromate solu- 
tion, remembering that, in these reactions, 11.2 milligrams of metallic iron are equivalent 
to 11.8 milligrams of metallic tin. It is convenient to use the same number of cc. for 
standardizing the stannous chlorid solution with drchromate as is used for titrating the 
methylene blue solution. 30.0 cc is a convenient amount in either case. It is to be borne 
in mind that these 30.0 cc do not represent exactly 11.8 milligrams of metallic tin, and 
that the exact amount of tin they represent is to be determined. The calculation will 
thus be made easy. 

No matter whether a dichromate solution is used or not, the strength of the methy- 
lene blue solution is always expressed by the number of cubic centimeters of the solution 
\vhich correspond to 11.8 milligrams of metallic tin. 

THE COLORIMETRIC STANDARDIZATION. 

In place of the chemical standardization an easier method may be used to determine 
the strength of a methylene blue solution. It may be done by simply comparing the 
intensity of the color of such solution with that of a solution of known strength. The 
blue color, in other words, shows the strength of the solution, the shade of the color 
being proportional to the strength. 

An exact comparison of the color of two solutions may be made by using two color 
comparison jars, known as Nessler jars. They are called Nessler jars from the fact that 
they are used extensively in sanitary water analysis for the determination of ammonia 
by the Nessler method. The best jars are those with polished bottom and uniform in 
clearness of glass and in shape. 

In comparing the colors of two methylene blue solutions, the two jars are set on a 
small, flat mirror placed horizontally on a well illuminated table. If equal volumes of 
the two solutions are found by comparison to have different intensities, or shades, of 
color, the volume of the stronger solution is decreased by pouring out some of it. The 
colors are then compared again. If the stronger solution is still too dark, a little more 
is poured out until the colors of the two solutions are exactly equal in intensity. When 
this is done, the height of the column of the stronger solution compared with the height 
of the column of the weaker solution shows how much stronger the former solution is 
than the latter and how much distilled water has to be added to it to make it equal in 
strength to the latter. 

This test may be made correctly by any intelligent person from the directions 
given. The titration with stannous chlorid, on the other hand, should not be attempted 
by anybody who has not had a college course in quantitative chemical analysis, for it 
may lead to incorrect results in the hands of an inexperienced operator. 

For most purposes, the colorimetric standardization is quite sufficient as a substi- 
tute for the titration. It is to be remembered, however, that the brands of methylene 
blue from which the two solutions are made, must be exactl}' the same. If the operator 

17 



is not sure that they are the same, he shouUl not apply the color comparison test or at 
least not rely on it. If, on the other hand, the two brands are the same, correct and 
fairly accurate results are obtainable by this test. It is inadvisable for any operator to 
use any brand of methylene blue other than that specified. 

Frequently, the need arises of comparing a freshly prepared solution with an old 
solution. For instance, in the spring of the year, a solution may have to be made of 
exactly the same strength as the solution used in the spring of the preceding year. In 
this case it is absolutely necessary to see to it that the old solution has not lost any 
of its strength. If a solution is kept in a tightly stoppered bottle filled to the top, in a 
warm room, it will lose its color completely in the course of a few months. To prevent 
such a decolorization, either partial pr complete, the solution is kept in bottles which are 
only half filled. The air re-oxidizes the solution. Furthermore, the sample of the solu- 
tion which is to serve for color comparison the succeeding year, is preserved with cor- 
rosive sublimate besides being kept in a half filled bottle. A one grain tablet of corrosive 
sublimate of the kind manufactured by Eli Lilly & Co., Indianapolis, and by similar firms, 
is quite sufficient to preserve 250 or 300 cc of solution for many years. The white tablets 
are to be used, not those having an admixture of dye stufif in them. 

Great care is to be taken that the corrosive sublimate is added only to those samples 
of solution which are to be preserved for color comparison, not to the ordinary testing 
solution which is added to milk samples. Although all the milk samples tested are 
wasted, a powerful chemical like corrosive sublimate should never be mixed with milk 
under any condition. The preserved solution should be kept locked up so it cannot be 
mistaken for ordinary testing solution. A poison label should be put on the bottle. The 
ordinary testing solution need not be preserved in that way. It retains its full strength 
several months, if it is kept in contact with air. even without a preservative. 

While a solution of known strength is necessary to make the color comparison, no 
such solution is required in the titration with stannous chlorid. Furthermore, the titra- 
tion with stannous chlorid need not be limited to any particular brand of methylene blue, 
as is the case with the color comparison. It can be applied to any kind or to any brand 
of methylene blue. The titration, therefore, has certain advantages. At the same time, 
it may be carried out without difficulty, by an experienced chemist. 

The three methods of standardization above described, will. I should think, be quite 
sufficient to eliminate completely the serious errors which were formerly brought about 
by the lack of any method of standardization and other defects in the solution. Other 
improvements consist in the modification of the form of the test. 

THE DOUBLE TIME TEST. 

The directions for this test are printed in bold type in order to attract attention. 
While this form of the color test is very simple, it is nevertheless important. It will he 
found useful in most cases where milk or cream are to be tested for bacteria and is well 
suited for general adoption. This is the reason why special attention is called to this 
test. 

If the color test is made with 8 (eight) cubic centimeters of methylene blue solu- 
tion instead of 2 (two) cubic centimeters, the time required for decolorization amounts 
to about double the time required with 2 cubic centmeters. This is the reason why this 
test, made with 8 cubic centimeters, is called "the double time test". 

This test offers two important advantages. It makes the blue color and the color 
changes much more conspicuous and it affords more accurate results. The disadvan- 
tage consists in that it takes twice as long to learn the results of the test, which, how- 

18 



ever, is not a serious drawback, except in certain cases. In the case of Grade B milk, 
or ordinary market milk, raw, the time required for making the test never amounts to 
more than eight hours and generally much less. Therefore, all such milk should be tested 
by the double time test. Likewise all raw cream. 

Grade A milk or infant milk, raw, on the other hand, would require too long a time 
with the double time test. Therefore this kind of milk or any similar milk should be 
tested with only 2 cubic centimeters of blue solution, in other words, by the single time 
test. 

Furthermore, a bacteriologist who uses the color test preliminary to plating for the 
purpose of determining what dilution he has to make, cannot afford to wait long enough 
for the results of the double time test. Therefore, he will do better to use only 2 cubic 
centimeters of the blue solution. 

The double time test is made in exactly the same way as the single time test where 
only 2 cubic centimeters of blue solution are used. Having however 8 cubic centimeters, 
therefore four times as much, blue solution to mix with the milk sample, it is absolutely 
necessary to insert the rubber stopper and to shake the tube thoroughly each time. 
Otherwise the color is not distributed uniformly and gives incorrect results. 

In the double time test as well as in the single time test, a blank tube must be put 
into the test tube holder along with the other samples. This blank tube contains only 
plain milk, without any blue solution whatever. The white color of this milk is com- 
pared with the color in the other tubes when they approach decolorization in order to 
determine whether there is a little blue color left or not. The exact time of decoloriza- 
tion can be determined more accurately that way. 

In the double time test as well as in the single time test all samples must be wasted 
after the test is completed and the time is noted down. Not only those samples must be 
wasted which are still blue, but also those which have been decolorized. 

As the time of the test is doubled when 8 cubic centimeters of blue solution are 
used instead of 2 cubic centimeters, the number of bacteria is estimated as follows : 

Less than Yi hour over 5 million 

1 hour 3 to 5 million 

13/ hours 2 to 3 million 

3 hours 1 to 2 million 

5 hours 300.000 to 1 million 

7 hours 100,000 to 300,000 

over 8 hours less than 200,000 

The results obtained in this manner are quite accurate and may be relied on as in- 
dicating correctly the extent of the bacterial activity in the milk. If, however, it be 
desired to get still more accurate results, this can be accomplished by observing the 
color of the tubes every half hour instead of every hour and reporting the results ac- 
cordingly. 

Milk dealers are advised to have their employees report merely the time required 
for decolorization and not the number of bacteria. As the time required for decoloriza- 
tion is quite sufficient to indicate the sanitary quality of the milk, it is unnecessary to 
figure out the number of bacteria each time, and besides, it is liable to lead to con- 
fusion with men who have had no experience in doing it. It is, however, necessary to 
state whether the double time test with 8 cubic centimeters of milk or the single time 
test with only 2 cubic centimeters of milk has been used. 

19 



It is advisable to standardize the methylene blue solution for the double time test 
separately and to keep a separate solution for this test. However, if a separate solution 
is not available, the solution prepared for the single time test may be used or vice versa. 
As the strength of the two solutions is almost the same, no serious harm is done by 
substituting one for the other. 

Raw cream may be tested in exactly the same way as raw milk. 

THE REDUCTASE NUMBER. 

While the number of live bacteria is indicated by the amount of reductase formed 
by them during the fermentation, at 112° F. (about 45° C), the total number of bacteria, 
including the dead, is indicated by the amount of reductase present in a sample of milk 
without any such fermentation. In other words, the amount of reductase found in milk 
samples without incubation or fermentation, or previous to such fermentation, is approx- 
imately proportional to the number of bacteria as determined by the direct microscopic 
count after Brew's or Skar's method or a similar method. As some bacteriologists re- 
gard the direct microscopic count, which includes the dead bacteria, as a better index of 
the sanitary quality of milk than the determination of the live bacteria by plating, more 
especially in the case of raw milk to be pasteurized, where the live bacteria are killed 
anyway by subsequent pasteurization, it seems desirable to adopt some form of the re- 
ductase test capable of giving about the same results as are obtained by the direct micro- 
scopic count. 

Accordingly, the total number of bacteria, including the dead, may be indicated 
rapidly by a direct determination of the reductase in a sample of raw milk without 
any fermentation. This can be done either by measuring the amount of blue solution 
decolorized within one minute, by a unit amount of milk, or by measuring the amount of 
milk necessary to decolorize, within one minute, a unit amount of blue solution. The 
latter method is the better, because the change of color is more distinct than in the other 
method, and because the large volume of milk can be measured more easily than the 
small volume of solution. 

The method is carried out as follows : 

1.0 cc of methylene blue solution of such strength that 100.0 cc are required in the 
stannous chlorid titration, for 11.8 milligrams of tin, in a suitable, graduated tube, is 
put into the water bath of the color test apparatus kept at 112° F. (about 45° C). A 
tubeful of the milk under examination is also put in. After both tubes have taken the 
temperature of the bath, the milk is added gradually to the blue solution and mixed with 
it until the blue color has completely disappeared. It is highly desirable to use a blank - 
tube with plain, uncolored milk for comparison of colors, so as to recognize complete 
decolorization. The number of cc of milk required is the "reductase number". 

A low reductase number indicates a higli uinnlnT of liacteria, while a high reduc- 
tase number indicates a low number of bacteria. 

With ordinary, raw market milk, the reductase number should not fall belnw 30, 
with raw infant milk, not below 50. 

This test is found useful to indicate excessive numbers of bacteria, dead and alive, 
in cases where the operator has no time to wait for the result of the regular color test, 
.more especially in summer. 

Apparently, the reductase number is a correct measure of the extent of that decom- 
position in milk which is liarmful to infants, in the case of raw milk to be pasteurized. 
While no particular accuracy is claimed for this test, it is quite sufficient to detect ex- 
cessive decomposition, which is a material advantage. 

20 



THE METHYLENE BLUE SOLUTION OF STANDARD STRENGTH. 

Instead of varying the strength of the methylene blue solution to conform to the 
plating results, and leaving the amount of the solution constant, the amount may be 
varied and the strength left constant. For instance, instead of using 2 cc of a solution 
of which 80 cc correspond to 11.8 milligrams of tin, in the stannous chlorid titration, 
2^ cc of a solution of which 100 cc correspond to 11.8 milligrams of tin, may be used. 
Likewise, instead of using 2 cc of a solution of which 133.3 cc correspond to 11.8 milli- 
grams of tin, I'j cc of a solution of which 100 cc correspond to 11.8 milligrams of 
tin, may be used. In each case, it will be noticed, a solution of the same strength, which 
is called "standard solution", may be used without altering the quantity of the dissolved, 
previously solid methylene blue, which is added to the milk sample or the result of the 
test, so long as the amount of the solution added is varied accordingly. If the standard 
solution is weaker than the solution to be replaced, it is evident that more of it has to 
be taken than was taken of the strong solution. If the standard solution is stronger, 
less of it is to be taken. 

In this way, the advantage is secured of having to use only one kind of solution 
under any conditions, for any kind of milk, for any form of the test, the double time test 
as well as the single time test, and at any season of the year, viz. : the standard solution. 

However, the operator who makes the tests must have proper appliances for measur- 
ing the varying amounts of the standard solution which may become necessary under 
varying conditions. For this purpose it is best to use short nozzle pipettes of the follow- 
ing capacities: 

For the single time test : 

In the winter 1 3^ cc 

In the spring 2 cc 

In the summer 2 J4 cc 

In the fall 2 cc 

For the double time test: 

In the winter 8 cc 

In the spring 10 cc 

In the summer 12 cc 

In the fall 10 cc 

If these quantities of the standard solution are used as stated, the results obtained 
will be at least approximately correct without any further adaptation to plating results. 
More accurate results, however, can be secured by checking the results with plating 
results, as directed under the heading "Bacteriological Standardization," with this dif- 
ference, however, that the plating results, which, in the other method, served to regulate 
the strength of the blue solution, are now used to regulate the amount of the solution 
instead. If, for instance, 10 cc of the standard solution, in the double time test, are 
found to give too high results, the amount of the standard solution is increased to 12 cc, 
or even, if necessary, to 15 cc. If 10 cc of the standard solution are found to give too 
low results, the amount is decreased to 8 cc or even to 5 cc. 

Likewise, in the single time test, the amount of the standard solution is to be in- 
creased from 2 cc to 2'j cc or even to 3 cc, or it may be decreased to i;i cc or even 
to 1 cc, as the plating results may require. 

It is difficult and unnecessary to tell beforehand how much more standard solution 
a given sample of milk will require in the double time test than in the single time test. 

21 



Some milk supplies require 5 times as much, while others require only 4 times as much. 
Irrespective of this ratio, the capacity of the pipettes is to be determined by plating 
results. 

In making the double time test with a standard solution, all the directions given for 
this test are to be carried out with the only exception that not always 8 cc of the blue 
solution are used, but either more or less, as the season or the plating results may 
require. 

In making the single time test with a standard solution, all the directions given for 
this test are to be carried out, with the only exception that not always 2 cc of the blue 
solution are used, but either more or less, as the season or the plating results may 
require. 

Attention is once more called to the fact that, when the standard solution is em- 
ployed, the solution used for the double time test is exactly the same as that used for 
the single time test, the strength being standard strength in either case. 

Whenever a standard solution is used, the chemical or colorimetric standardization 
precedes the bacteriological checking of the color test results. By "standard" solution 
is meant a solution of which exactly 100 cc are required for 11.8 milligrams of tin, in the 
titration with stannous chlorid. Therefore, in preparing a "standard solution", this is 
the strength to be given to the solution. 

Additional solutions of equal strength may then be prepared, at any time there- 
after, by the colorimetric test, so long as the same brand of methylene blue is used, or 
the titration with stannous chlorid may again be employed to standardize such solutions. 

After the standard solution is furnished for the color test, the plating tests on agar 
are made to determine how many cc. of the solution are to be used for any given milk 
supply, as stated, without changing the strength of the solution. 

Under ordinary conditions, the standard solution offers greater advantages than the 
solutions of variable strength. It is therefore to be preferred. However, under unusual 
conditions, it is possible that the solutions with variable strength may give better 
satisfaction, for the reason that they obviate the necessity of changing pipettes. As 
creameries sometimes employ men who do not know the first thing about laboratory 
work, wrong pipettes are liable to be used in some cases. To preclude the possibility 
of such an error, it may possibly be best to furnish only one pipette to such men, even 
if it puts the bacteriologist or chemist to the trouble of preparing three or four different 
solutions each year. 

As soon as the color test comes into general use, and is better understood, all 
creamery employees, including the less intelligent, will undoubtedly be able to use an 
entire assortment of pipettes correctly, and the exclusive use of the standard solution 
will then be in order. 



MATCHING EXTRAORDINARY PLATING RESULTS. 

The short nozzle pipettes of the capacities stated above will give about the same 
results as the plating tests, if the plating is done on standard infusion agar and the count- 
ing with the naked eye. If, on the other hand, beef extract agar containing 1.0 per cent 
of powdered, dry agar and 0.6 per cent of normal acid, is used, and the counting is done 
with a lens of 3'^ diameters magnification, short nozzle pipettes of the following cap- 
acities will deliver the proper amount of standard methylene blue solution to match the 
plating results : 

11 



For the single time test: For the double time test: 

In the winter 1 cc. In the winter 5 cc. 

In the spring 15^ cc. In the spring 7 cc. 

In the summer 2 cc. In the summer 9 cc. 

In the fall 1>4 cc. In the fall 7 cc. 

It is understood that the dilutions of the milk in the plating are such that not over 
200 colonies develop on each plate. If considerably more develop, the counts are incor- 
rect and the color test results can, therefore, not be expected to agree with them. 

Furthermore, as near sighted persons get higher results than far sighted persons, in 
counting agar plates, the color test results cannot be expected to agree with the plating 
results, unless the counting is done by a person having normal eyes, or wearing correct- 
ing eye glasses. Likewise, on a bright day, the plating results run higher than on a 
cloudy day, since the small colonies are overlooked on a cloudy day. This is another case 
where the plating results, being incorrect, cannot always be matched by the color test. 

Moreover, as rapidly growing bacteria decompose the milk more rapidly than others, 
the plating test fails to indicate correctly the extent of decomposition which the milk has 
undergone, since the small colonies formed by slowly growing bacteria are counted just 
the same as the large colonies formed by rapidly growing bacteria. The color test, for 
obvious reasons, indicates much better the extent of the decomposition and, therefore, it 
cannot be expected to agree with the results of the plating test, which, in the case of 
raw milk to be pasteurized, are misleading. This applies especially to cases where there 
are many small colonies on an agar plate, or else many large colonies. 

That it would be a big mistake to condemn the color test merely because it some- 
times fails to give the same results as the plating test, is most evident, I should think. 
\\'ith the plating test, errors are almost inevitable, but not with the color test. 

PROPOSED OFFICIAL MODIFICATION OF THE COLOR TEST. 

As soon as official limits are adopted on the color test, it will no longer be expected 
to follow the erratic fluctuations of the plating test. Color test results will have a mean- 
ing of their own, independent of the plating test. Their value will no longer be ob- 
scured by comparison with that test. It will then no longer be necessary to use different 
pipettes in the different seasons or otherwise to adapt the color test to the plating re- 
sults. A 2 cc. pipette could be used for the single time test at any time of the year, and 
an 8 cc. pipette for the double time test, also at any time of the year. All official limits 
could be based on tests made with these two pipettes, with standard methylene blue 
solution, at a temperature of 112° F. While it is possible to use a lower temperature, it 
does not seem desirable for the reason that low temperatures would bring out excessive 
numbers of the lactic acid bacteria and would generally fail to bring out those bacteria 
which are foreign to milk. The amount of standard solution, the temperature, and the 
permissible time of decolorization are to be specified with any official limits which may 
be adopted, since without these specifications, the limits would be indefinite. The vary- 
ing conditions of the seasons, climatic conditions, and the distance of the milk producing 
districts from the city, these matters want to be taken into consideration in fixing official 
limits. The limits need not necessarily be the same for different cities. Furthermore, a 
summer standard and a winter standard is recommended for each city. The former, 
however, is naturally much more important than the latter. 

The general official and private adoption of the color test is sure to come, for it will 
be found to afford very valuble results at a moderate expense. One operator can 
sample and test over 120 different lots of milk in 8 hours, while the ]ilating tost requires 
much more time. The color test will sa\-e thousands of infants. 

23 



THE COLOR TEST WITH EXCLUSION OF AIR. 

In the "Directions" it was stated that those tubes which had a blue ring at the top 
were to be regarded as decolorized so long as the rest of each tube was white. In doing 
^o, correction is made for the blue color re-formed from previously decolorized methylene 
li'ue by absorption of oxygen from the air. Instead of making this correction, the re- 
formation of the blue color may he prevented altogether by excluding the air. This can 
be accomplished by using 30 cc of milk instead of 20 cc, heating to 112° F., as usual, 
adding the regular amount of methylene blue, dropping in enough sterilized glass beads 
to raise the level of the liquid to a point about half an inch from the top of the tube, and 
finally displacing the last remaining air space by inserting a perforated cork with a 
short, narrow glass tube in the centre to permit the escape of the air. The mixing of the 
blue solution with the milk is effected in the absence of air by slanting the tube in such 
a way that the glass beads drop from the bottom of the tube to the top and back again 
6 or 8 times. The opening of the glass tube may be closed with a clean finger during 
the mixing. The test tube which is evidently' brim full and deprived of air, is placed in 
the water bath as usual. As the growth of the aerobic organisms is restricted under 
these conditions, the decolorization requires about the same length of time with 30 cc of 
milk as it would with only 20 cc of milk and the same amount of methylene blue solu- 
tion when in contact with air. For this reason, no more blue solution is used with the 
30 cc of milk than with 20 cc in the other form of the test, and the number of bacteria is 
estimated by the same tables. It is necessary, however, to add an "intermediary" test 
and table using twice as much blue solution as in the single time test, because the color 
is too faint if the single time test is used with 30 cc of milk instead of 20 cc. Ordinarily, 
4 cc of blue solution are used with 30 cc of infant milk, raw, and 8 cc of blue solution 
with 30 cc of common market milk, raw, unless a desire to match the plating results 
under abnormal conditions requires a modification of these amounts. 

The Intermediary Test, requiring irom 2 cc to 6 cc of standard methylene blue 
solution, as the plating results may indicate : 

Time required Number of Time required Number of 

for decolorization : bacteria : for decolorization : bacteria : 

Less than 23 niin over 5 million 3 hours '/< to IjX million 

34 hour 3 to 5 million 4 hours 200,000 to 800,000 

1 hour 2 to 3 miKion 5 hours 100,000 to 300,000 

2 hours 1 to 2 million over 6 hours less than 200,000 

The corks and the glass beads, which should be ^ to J4 inch in diameter, are to be 
cleaned and sterilized after each test along with the test tubes. The cotton stoppers 
and the solid ruliber stopper are no longer needed when the corks are used. In exact 
work, it is desirable to use sterilized glass beads not merely for the purpose of displacing 
the air and mixing the blue solution with the milk, but also for breaking up clumps in 
the uncolored sample before the 30 cc are removed. The beads are shaken with the 
sample, which must be cold, 25 or 30 times. They must evidently be strained out later" 
on, if any part of the sample is to be used for human consumption. Before the color is 
added, each sample should stand in the water bath 5 minutes to get warm and rid of air 
bubbles. There should be sufficient water in the bath to keep the tubes warm all the 
way from top to bottom. With milk samj^les which contain sediment, great care is to be 
taken to have the sediment distributed uniformly throughout the entire sample. Other- 
wise, incorrect results are obtained with any kind of test. 

By e.xcluding the air in the color test, miiform results are obtained because the 
amount of oxygen absorbed no longer varies. This form of the test is therefore to be 
preferred in all cases except where the operator is incompetent to use the glass beads 
and the corks without infecting the sample. Such operators may continue the air space 
in the tubes and get the best results possible for them by carefully avoiding any further 
agitation of the sample after the blue color has been mixed with it. Such agitation would 
evidently cause absorption of oxygen and spoil the results. As a preliminary test for 
plating, the "intermediary" test with 4 cc of blue solution, and with exclusion of air, is 
l^etter than any other test. The blue solution is to be kept in an ice box. 

24 



The Apparatus 

and the Standard Solution for the Color Test 

are kept for sale by the following dealers in 

bacteriological and chemical supplies: 



ElMER & AMEND, 

205 to 211 Third Avenue. 
NEW YORK CITY. 



BAUSCH & LOME" OPTICAL CO., 

ROCHESTER, N. Y. 



E. H. SARGENT & CO., 

125-127 W, Lake Street, 
CHICAGO, ILL. 



LIBRftRY OF CONGRESS 



000 895 575 fl f 



1 



